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Sample GSM4202920

Query DataSets for GSM4202920

Status

Public on Dec 05, 2019

Title

JunB.ECKO.rep1.431

Sample type

SRA

Source name

RNA.JunB.ECKO

Organism

Mus musculus

Characteristics

strain background: C57BL/6
genotype/variation: Junbflox/flox Cdh5-CreERT2
age: postnatal day 6
cell type: FACS-sorted adult mouse retinal endothelial cells

Treatment protocol

"Control" and "gTKO" pups were given intraperitoneal injections of tamoxifen (100 ug) from P1-P3. "JunB.Control" and "JunB.ECKO" pups were given subcutaneous injections of 4-hydroxytamoxifen (50 ug) from P1-P3.

Growth protocol

Mice were housed in our animal facility with food ad libitum and 12 hr light/dark cycles. Animals were used in accordance with protocols approved by the Institutional Animal Care and Use Committees (IACUC) of Boston Children’s Hospital and the French Department of Education.

Extracted molecule

polyA RNA

Extraction protocol

bulk RNA-seq: Cells were sorted into either 0.1% FAF-BSA/PBS or buffer RLT (Qiagen) supplemented with beta-mercaptoethanol for ATAC-seq and RNA-seq, respectively. Cells sorted into buffer RLT were subjected to total RNA extraction using the RNeasy Micro Kit (Qiagen).
ATAC-seq: ATAC-seq libraries were prepared according to the previously described fast-ATAC protocol (Corces et al., 2016). Briefly, 5,000-8,000 FACS-isolated cells in 0.1% FAF-BSA/PBS were pelleted by centrifugation at 400 xg at 4 ˚C for 5 min. Supernatant was carefully removed to leave the cell pellet undisturbed, then cells were washed once with 1 mL ice-cold PBS. The transposition mix [25 µL buffer TD, 2.5 µL TDE1 (both from Illumina FC-121-1030), 1 µL of 0.5% digitonin (Promega, G9441) and 16 µL nuclease-free water] was prepared and mixed by pipetting, then added to the cell pellet. Pellets were disrupted by gently flicking the tubes, followed by incubation at 37 ˚C for 30 minutes in an Eppendorf ThermoMixer with constant agitation at 300 rpm. Tagmented DNA was purified using the MinElute Reaction Cleanup Kit (Qiagen, 28204), and subjected to cycle-limiting PCR as previously described (Buenrostro et al., 2013). Transposed fragments were purified using the MinElute PCR Purification Kit (Qiagen, 28004) and Agilent DNA Tapestation D1000 High Sensitivity chips (Agilent) were used to quantify libraries.
RNA-seq: Double-stranded cDNA was synthesized from 5-10 ng RNA using the SMART-Seq2 v4 Ultra Low RNA Kit for Sequencing (Takara) according to the manufacturer’s instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

JunB.ECKO.rep1.431
processed data file:
GEO_JunB.RNAseq_edgeR.and.RSEM_FPKMs.xlsx
JunB.ECKO.428.429.431.merged.RNA.bw.gz

Data processing

ATAC-seq: Read alignment to the MGSCv37 (mm9) genome assembly was performed with bowtie2 (Langmead & Salzberg, 2012) and the options: --very-sensitive –X 2000 –no-mixed –no-discordant. Duplicated fragments were removed using the Picard “MarkDuplicates” script with the options: Remove_Duplicates=true Validation_stringency=lenient (http://broadinstitute.github.io/picard/). Paired-end reads were separated, centered on Tn5 cut sites, and trimmed to 10 bp using a custom in-house script. Peaks were called using the MACS2 “callpeak” script (Zhang et al., 2008) with options: -B –keep-dup all –nomodel –nolambda –shift -75 –extsize 150. Reads mapping to murine blacklisted regions and mitochondrial DNA were masked out of peak lists using the Bedtools “intersect -v” script (Quinlan & Hall, 2010). Replicates from each biological group were merged using the bedops “merge” script to generate one high-confidence peak set for each of the two biological groups (Control and gTKO) (Neph et al., 2012). These two peak sets were then merged to generate a final merged peak set of 62,748 peaks. For each replicate, reads covering consensus peak intervals were counted using the Bedtools “coverage” script with the “-counts” option (Quinlan & Hall, 2010). The resultant count table was input to edgeR (M. D. Robinson et al., 2010) to determine differentially accessible peaks (DAPs), which can be found in the excel file “GEO_retina_ATACseq_S1PR.gTKO_edgeR.xlsx”. HOMER results from different peak inputs are in the excel file “GEO_retina_ATACseq_HOMER_results.xlsx”.
Base calls were performed using Illumina CASAVA 1.8.2
bigWig files: Nucleotide-resolution coverage (bigWig) tracks were generated by first combining trimmed reads from each replicate, then inputting the resultant .bam files to the DeepTools (Ramirez et al., 2016) “bamCoverage” script with options “—normalizeUsing RPGC –binSize 1”.
bulk RNA-seq: Reads from each sample were aligned to the MGSCv37 (mm9) genome assembly using STAR (Dobin et al., 2013) with the options: --runMode alignReads --outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --alignIntronMin 10 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outSAMtype BAM SortedByCoordinate --quantMode TranscriptomeSAM. Gene-level counts over UCSC annotated exons were calculated using the Rsubread package and “featureCounts” script (Liao, Smyth, & Shi, 2013) with options: -M –O –p –d 30 –D 50000. The resultant count table was input to edgeR (M. D. Robinson, McCarthy, & Smyth, 2010) for differential gene expression analysis.
bulk RNA-seq: The .bam files from STAR were input to the RSEM (B. Li & Dewey, 2011) script “rsem-calculate-expression” with default parameters to generate FPKMs for each replicate. FPKMs are located in the excel files called "GEO_JunB.RNAseq_edgeR.and.RSEM_FPKMs.xlsx" and “GEO_Retina_S1PR.gTKO.edgeR_RNAseq.xlsx”.
Genome_build: mm9
Supplementary_files_format_and_content: The excel files "GEO_JunB.RNAseq_edgeR.and.RSEM_FPKMs.xlsx" and “GEO_Retina_S1PR.gTKO.edgeR_RNAseq.xlsx” include RSEM-generated FPKMs and the edgeR output for RNA-seq experiments. The excel file “GEO_retina_ATACseq_S1PR.gTKO_edgeR.xlsx” includes the edgeR output for the ATAC-seq experiment. HOMER results from different peak inputs are in the excel file “GEO_retina_ATACseq_HOMER_results.xlsx”. All 58,311 and 50,178 merged Control and gTKO peaks, respectively, are included in the .bed files “retina.Control_peaks.narrowPeak.bed”, and “retina.gTKO_peaks.narrowPeak.bed”. The bigWig files were generated as described in "data processing".

Submission date

Dec 04, 2019

Last update date

Dec 06, 2019

Contact name

Eric Engelbrecht

E-mail(s)

eric.g.engelbrecht@gmail.com

Phone

9143917079

Organization name

Boston Children's Hospital